1. Field of the Invention
The present invention relates to a useful deoxyribonuclease as a reagent for gene manipulation and to a process for producing said enzyme using genetic engineering techniques.
2. Description of the Related Art
The enzymes most frequently used as deoxyribonucleases that recognize specific sequences and cleave double-stranded DNAs (that is, genes), base sequence-specifically, are those called restriction enzymes, and they typically recognize the sequences of 4 to 8 bases. Restriction enzymes have played essential roles in genetic engineering experiments: they have been used in daily gene manipulation experiments, and thereby greatly contributed to advances in molecular medicine, molecular biology, and biochemistry.
In addition to restriction enzymes, as enzymes that recognize nucleotide sequence and cleave double-stranded DNAs, there is a group of enzymes called homing endonucleases, which are involved in the DNA recombination process. Although these enzymes generally require sequences as long as 20 or more bases for recognition, recognized sequences are specific for respective enzymes, and therefore, these enzymes can be used for the purpose of site-specific DNA cleavage.
Thus, there have been many practical applications of enzymes that recognize and cleave DNA sequences. As to enzymes that recognize and cleave specific steric structures of DNA, however, only few studies are known with particular emphasis on Escherichia coli RuvC protein, and there are no enzymes that have already come into practical use, although biochemical properties such as substrate specificity have been elucidated to some extent.
Although several enzymes that recognize and cleave a specific steric structure such as the Holliday structured are presently known, they are all derived from mesophilic organisms, and their thermostability or cleavage efficiency in vitro is low. An object of the present invention is to develop a practical enzyme that specifically recognize and cleave a Holliday structured DNA, which is a DNA recombination intermediate, to resolve it into two sets of double-stranded DNAs, and to provide such enzyme as a reagent for gene manipulation.